This invention describes a method for identifying bacteria. In particular, this invention relates to a method for identifying and quantifying mycobacteria from a sputum sample using flow cytometry. Further described is the use of flow cytometry to identify and quantify Mycobacterium tuberculosis from sputum-derived samples for verification, calibration and/or the establishment of a quality control system for use during diagnosis of tuberculosis (TB).
Despite the identification of M. tuberculosis as the cause of TB more than a century ago, diagnosing TB in resource poor countries remains a challenge, especially in people living with HIV. TB is the disease caused by infection of M. tuberculosis, which remains an important cause of morbidity and mortality, with over 9 million new cases and about 1.8 million deaths annually. Due to the contagious nature of the disease, rapid identification is crucial to minimise the transmission rate between individuals in communities and health care facilities.
The most common method for diagnosing TB in most countries is smear microscopy, which is cheap and relatively easy to perform, but has modest sensitivity (35 to 80%). The sensitivity of smear microscopy is especially poor in HIV-infected individuals and children, populations that comprise the majority of cases in many sub-Saharan African countries. The accepted gold standard for TB diagnosis is culture for M. tuberculosis. This method is highly sensitive and allows identification of M. tuberculosis and differentiation between drug-sensitive and drug-resistant strains, but it has many limitations. The greatest limitation is the turnaround time of 2 to 6 weeks, which reduces its usefulness in clinical decision-making. Another limitation is that for culture to be performed, it has to be carried out in a level of biosafety infrastructure (BSL 2 to 3) with particular laboratory operational requirements, which cannot be implemented easily in decentralized resource poor settings. Even with the use of liquid culture methods such as BACTEC™ MGIT960 and MGIT DST (BD Diagnostic Systems) there are still shortcomings in that the methodology is still “human resource” intensive, costly and prone to contamination. There has therefore recently been acceleration in the development of new TB diagnostic methods, especially with the need to prevent TB infection in the wake of the HIV pandemic.
According to the StopTB Partnership (http://www.stoptb.org/), current and new technologies for active and latent TB diagnosis can be divided into their use in reference laboratories, peripheral laboratories and clinic based settings (POC), and consists of several different types of technologies. These are:                (i) Nucleic Acid Amplification Tests (NAAT) for Multiple Drug Resistant (MDR)-TB diagnosis and speciation assay such as GenoType® MTBDR and MTBDRplus (Hain LifeSciences), INNO-LiPA Rif.TB (Capilia TB-Neo), GeneXpert MTB/RIF (Cepheid), loop-mediated isothermal amplification technology (TB-LAMP, Eiken Chemical Co), COBAS TaqMan MTB (Roche Molecular Systems), Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (AMTD) and enhanced AMTF (E-AMTD, Gen-Probe), LightCycler Mycobacterium detection kit (LCTB) (Roche Applied Science), Seeplex TB Detection Kit (Seegene, Korea), BD-ProbeTec Direct and the semi-automated ProbeTec ET system (Becton Dickinson) and the Abbott LCx (Abbott Molecular).        (ii) Interferon Gamma Release Assays for diagnosis of latent M. tuberculosis infection (QuantiFERON®-TB Gold In Tube, T-SPOT.TB®) and the TB patch test also for the diagnosis of latent infection.        (iii) Antigen based assays such as lipoarabinomannan (LAM) and breathalyzer screening test for screening for TB.        (iv) Phage based tests for rapid diagnosis of MDR-TB.        (v) Other methods for detection of drug resistant M. tuberculosis: the calorimetric redox indicator method, Microscopic Observation Drug Susceptibility (MODS), nitrate reductase assay, and thin layer agar culture, bleach microscopy, sputum filtration, vital fluorescent staining of sputum stains for improved smear microscopy, light Emitting Diode (LED) fluorescence microscopy for improved direct smear microscopy.        
Studies of patients in the HIV prevalent areas of sub-Saharan Africa have shown that the risk of tuberculosis incidence is 8.3 times higher for those who are infected with HIV than for those who are uninfected. Smear-negative tuberculosis is more common amongst HIV-positive persons, and is difficult to diagnose. The WHO has established algorithms for the diagnosis of smear negative TB in HIV-positive persons. Sputum PCR offers the potential for improved sensitivity compared to TB microscopy, although some variability has been reported 0.85 (range 0.36-1.00) and specificity of 0.97 (range 0.54-1.00). A new PCR method from Cepheid called the GeneXpert MTB/RIF, however, offers great promise since the first reports of sensitivity and specificity on smear-positive culture positive patients of 98.2% and smear-negative culture-positive patients of 72.5%. Furthermore, a study conducted by the Department of Molecular Medicine and Haematology with Witwatersrand Reproductive Health and HIV Research Unit (RHRU) on outpatient pulmonary adult TB patients, 70% of which were coinfected with HIV, revealed that the sensitivity of a single GeneXpert MTB/RIF test compared to a single BACTEC MGIT culture was 86%. This method is a real-time PCR based system that requires 2 minutes hands-on-time and generates a result within 2 hours, which also includes sensitivity or resistance to rifampicin. This system can be performed at the bedside by ‘non-technically skilled’ persons and does not require biosafety infrastructure. Limitations of this method, however, are high cost and its current modular format, i.e. the instrument design is presented as modules of 1-16, 32 and 48. The modules are currently limited to low throughput bedside testing or small laboratories, and solutions for high throughput laboratory testing, especially needed in large centres where there is high HIV/TB prevalence, are still urgently needed. Despite these limitations, the World Health Organisation has endorsed the use of the GeneXpert MTB/RIF assay in TB endemic countries and declared it a major milestone for global TB diagnosis.
In order for an endemic country's health system to implement the GeneXpert MTB/RIF assay in response to global health principles, a number of aspects require attention for functional implementation of this assay. These include: determining the main logistical and operational issues related to GeneXpert MTB/RIF implementation (e.g., cartridge supply, downtime of the GeneXpert unit); establishing a sample database for calibration and verification of the results obtained for each GeneXpert instrument installed (before the instrument is deemed acceptable for reporting patient results); and generating appropriate external quality assessment (EQA) programs to ensure on-going quality testing in both laboratory and non-laboratory settings.
It is anticipated that based on the specific GeneXpert MTB/RIF assay characteristics, verification and EQA programs will require the following criteria: 1) testing material must contain whole M. tb for capture in the GeneXpert MTB/RIF cartridge; 2) transportation of EQA material needs to be safe; 3) the testing procedure needs to be safe and compatible with the GeneXpert MTB/RIF current testing protocol; non-laboratory skilled health care workers must be able to perform the testing in non-laboratory settings; and the programs will need to be cost effective and sustainable for national programs.
Apart from the technologies describe above and their associated shortfalls, the diagnosis of TB, via the identification and quantification of M. tuberculosis, in an HIV endemic area have several further complications and implications: HIV infection often leads to a reduction in sputum production and a reduction in good quality sputum. This leads to an increase in the number of smear negative culture positive cases. This in turn results in delayed diagnosis, which leads to an increased risk of transmission. In addition, diagnostic tests are required for screening of people living with HIV to aid in the eligibility assessment for preventive therapy, and diagnosis of TB in people eligible for antiretroviral treatment.
Therefore, the solution for TB diagnosis may become multifactorial in that clinical algorithms as well as applying different types of tests for different scenarios may be needed. In particular, there is a need for a rapid test for identifying the presence of M. tuberculosis in a sample taken from a patient (i.e. one which can provide a result within 24 hours) and which is relatively easy to perform and which is also affordable. Furthermore, there is a need to establish a reliable method, for use with the GeneXpert MTB/RIF assay, for generating a sample database for use in calibration and verification of the results obtained by this method and further for on-going external quality control assessment.